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Abstract:

Probiotic compositions comprising a bacterium that is capable of
producing nitric oxide and a substrate of nitric oxide synthase are
provided. Also provided are methods of improving the gastrointestinal
health of a subject by orally administering a bacterium capable of
producing nitric oxide and orally administering a substrate of nitric
oxide synthase. Methods of reducing the inflammatory immune response
after treatment with a probiotic and methods of reducing horizontal
transmission of pathogenic bacteria within groups of animals are also
provided.

Claims:

1. A probiotic composition comprising a bacterium and a substrate of
nitric oxide synthase, the bacterium capable of producing nitric oxide.

2. The probiotic composition of claim 1, wherein the substrate of nitric
oxide synthase is a nitrate, nitrite or salt thereof.

3. The probiotic composition of claim 2, wherein the nitrite or nitrate
concentration is between 10 and 10,000 ppm.

5. The probiotic composition of claim 1, wherein the composition is
formulated for administration in or with feed or water.

6. The probiotic composition of claim 1, wherein the composition further
comprises bacteria not capable of producing nitric oxide.

7. A method of improving the gastrointestinal health of a subject
comprising orally administering a bacterium capable of producing nitric
oxide to the subject and orally administering a substrate of nitric oxide
synthase to the subject, wherein administration improves the
gastrointestinal health of the subject as compared to a control.

8. The method of claim 7, wherein the gastrointestinal health is improved
by reducing the prevalence of pathogenic bacteria in the gastrointestinal
tract of the subject, reducing the incidence or severity of necrotic
enteritis, increasing the weight gain over time or improving the feed
conversion ratio as compared to a control.

9. The method of claim 7, wherein the substrate of nitric oxide synthase
is nitrate, nitrite or a slat thereof.

10. The method of claim 9, wherein the nitrate or nitrite concentration
is between 10 and 10,000 ppm.

11. The method of claim 9, wherein the nitrate or nitrite concentration
is between 50 and 1000 ppm.

12. The method of claim 7, wherein oral administration is in or with feed
or water.

14. The method of claim 7, wherein the composition further comprises
bacteria not capable of producing nitric oxide.

15. The method of claim 7, wherein the subjects are poultry.

16. The method of claims 7, wherein the bacteria and the substrate of
nitric oxide synthase are administered at the same time.

17. A method of reducing the inflammatory immune response after treatment
with a probiotic composition including bacteria capable of producing
nitric oxide comprising orally administering a substrate of nitric oxide
synthase to the subject, wherein administration of the substrate of
nitric oxide synthase reduces at least one immune effector as compared to
control animals.

18. The method of claim 17, wherein the immune effector is selected from
IL-2, IL-4 and IFN-.gamma..

19. The method of claim 17, wherein the substrate of nitric oxide
synthase is nitrate, nitrite or a salt thereof and the concentration is
between 10 ppm and 10,000 ppm.

20. The method of claim 17, wherein the subjects are poultry.

21. A method of reducing horizontal transmission of pathogenic bacteria
in a group of animals comprising orally administering a bacterium capable
of producing nitric oxide to the animal and orally administering a
substrate of nitric oxide synthase to the animal, wherein administration
of the bacterium and the substrate reduces the pathogenic bacteria in the
animal and thereby reduces horizontal transmission between animals in the
group.

22. The method of claim 21, wherein the substrate of nitric oxide
synthase is nitrate, nitrite or a salt thereof and the concentration is
between 10 ppm and 10,000 ppm.

23. The method of claim 21, wherein the subjects are poultry.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority of U.S.
Provisional Patent Application No. 61/506,981, filed Jul. 12, 2011, which
is incorporated herein by reference in its entirety.

[0002] The present invention relates in general to improving the health of
agricultural animals and reducing the chance of pathogenic bacterial
contamination of food products by animal waste or during the slaughter
process. In particular, probiotic formulations comprising bacteria and
methods of using the same to improve the health of domestic animals, in
particular poultry, are provided. The use of antibiotics in animal
agriculture, in particular poultry production, is coming under increasing
pressure from both consumers and government regulatory agencies.

[0003] This has created a need for effective antibiotic alternatives. The
use of probiotics or direct-fed microbials (DFM) in animal agriculture
may be one such potential alternative. The use of probiotics or DFMs may
reduce the use of antibiotics in agricultural animal production and
safeguard the food supply. In particular the use of probiotics or DFMs
may improve the gastrointestinal health, increase weight gain, improve
feed conversion ratios, and reduce the prevalence of pathogenic bacteria
in the gastrointestinal tract of the animals.

SUMMARY

[0004] Probiotic compositions or direct-fed microbials and methods of
using probiotic compositions to increase the health of subjects, such as
poultry, and reduce horizontal transmission to other animals and humans
are provided herein. In one aspect, probiotic compositions including a
bacterium capable Of producing nitric oxide and a substrate of nitric
oxide synthase, such as nitrate or nitrite, are provided. The nitrate or
nitrite may be provided such that the concentration in feed or water is
between 10 ppm and 10,000 ppm.

[0005] In another aspect, methods of improving the gastrointestinal health
of a subject are provided. The methods include oral administration of a
bacterium capable of producing nitric oxide and of a substrate of nitric
oxide synthase to the subject. The administration improves the
gastrointestinal health of the subject as compared to a control.

[0006] In yet another aspect, methods of reducing the prevalence of
pathogenic bacteria in the gastrointestinal tract of a subject are
provided. The methods include oral administration of a bacterium capable
of producing nitric oxide and a substrate of nitric oxide synthase such
as nitrate or nitrite to the subject. The administration reduces the
prevalence of pathogenic bacteria in the gastrointestinal tract of the
subject.

[0007] In still another aspect, methods of reducing horizontal
transmission of pathogenic bacteria in a group of subjects are provided.
The methods include oral administration of a bacterium capable of
producing nitric oxide and a nitric oxide synthase substrate such as
nitrate or nitrite to the subject. The administration reduces horizontal
transmission between subjects by reducing the pathogenic bacterial load
in the group of subjects.

[0008] In a further aspect, methods of reducing the inflammatory immune
response after treatment with a probiotic composition are provided.
Suitably the probiotic composition comprises bacteria capable of
producing nitric oxide. Alternatively bacteria capable of producing
nitric oxide may be administered to the subjects in addition to the
probiotic composition. In addition to the probiotic, a substrate of
nitric oxide such as nitrate or nitrite is orally administered to the
subject. Administration of the substrate of nitric oxide synthase is
capable of reducing at least one immune effector, such as IL-2, IL-4 or
IFN-γ production in the subject as compared to control subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 is a graph showing the effects of adding a probiotic and
various concentrations of nitrate on the growth of Salmonella enteritidis
in vitro.

[0010]FIG. 2 is a graph showing the effects of adding a probiotic and
various concentrations of nitrate on the growth of Salmonella enteritidis
in vivo in either the crop or the cecal tonsils at 24 hours after
treatment.

[0011]FIG. 3 is a graph showing the effects of adding a probiotic and
various concentrations of nitrate on the growth of Salmonella enteritidis
in vivo in tither the crop or the cecal tonsils at 72 hours after
treatment.

[0012]FIG. 4 is a graph showing the effects of adding a probiotic and
optionally 100 ppm of nitrate on the growth of Salmonella enteritidis in
vivo in either the crop or the cecal tonsils at 24 hours after treatment.

[0013] FIG. 5 is a graph showing the effects of adding a probiotic and
optionally 100 ppm of nitrate on the growth of Salmonella enteritidis in
vivo in either the crop or the cecal tonsils at 72 hours after treatment.

[0014]FIG. 6 is a graph showing the effects of adding a probiotic and
optionally 100 ppm of nitrate on the IL-4 mRNA levels in the ceca in vivo
at 72 hours after treatment.

[0015] FIG. 7 is a graph showing the effects of adding a probiotic and
optionally 100 ppm of nitrate on the IFN-γ mRNA levels in the ceca
in vivo at 72 hours after treatment.

[0016]FIG. 8 is a graph showing the effects of adding a probiotic and
optionally 100 ppm of nitrate on the IL-2 mRNA levels in the ceca in vivo
at 72 hours after treatment.

DETAILED DESCRIPTION

[0017] Recently, we determined that some of the key bacterial isolates in
a commercially available probiotic, FloraMax-B11®, are capable of
producing the powerful antimicrobial compound nitric oxide (NO). As
nitrate substrates may be able to serve as precursors for NO synthesis by
bacterial cells, we postulated that incorporation of nitrate or another
nitric oxide synthase substrate into the medium, in the case of in vitro
tests, or into chicken feed for in vivo testing, would increase the
efficacy of this product on reducing pathogens such as Salmonella spp.
The data provided in the Examples below describe the improved efficacy of
this product in the presence of an added substrate of nitric oxide
synthase both in vitro and in vivo.

[0018] Nitric oxide, a free radical gas, produced by phagocytes and other
immune system cells has been shown to, have immunomodulating and
antibacterial effects. As described in the Examples below, we found that
a combination of the bacteria in the FloraMax-B11® probiotic and
addition of nitrate, a substrate of the nitric oxide synthase, reduced
the ability of Salmonella enteritidis to replicate both in vitro and in
vivo. The results suggest that administering bacteria capable of
producing nitric oxide in combination with a substrate of nitric oxide
synthase, in particular a nitrate or nitrite, can increase the
gastrointestinal health of animals and reduce the pathogenic bacterial
growth or load in the subject's gastrointestinal tract. In addition, we
expect that horizontal transmission of pathogenic bacteria from one
animal to another within a group of animals would be reduced and that
reduced pathogenic bacterial loads in animals will result in reduce
contamination of animal-based food products including meat and eggs.

[0019] The methods may be carried out by orally administering a bacterium
capable of producing nitric oxide and a nitric oxide synthase substrate,
such as nitrate or nitrite to the subject. Oral administration can be by
any known method including oral gavage, ingestion in feed or water or via
any other means available to those of skill in the art. The bacteria
capable of producing nitric oxide and the substrate of nitric oxide
synthase can be administered in a single probiotic composition or may be
administered separately. The bacteria may be administered before, at the
same time or after administration of the substrate of nitric oxide
synthase. The bacteria and the substrate of nitric oxide synthase may be
administered in a single dosage form or may be administered continuously
in the feed or water. In one embodiment, the bacteria are administered in
a single dose and the substrate of nitric oxide synthase is provided
continuously in the feed or water.

[0020] Bacteria capable of producing nitric oxide are known to those of
skill in the art or can be determined using available tests for nitric
oxide production. FloraMax-B11® contains several lactic acid bacteria
capable of producing nitric oxide. Lactic acid bacteria or other bacteria
capable of producing nitric oxide other than those in the
FloraMax-B11® probiotic may be used in the compositions, and methods
described herein. The bacteria may be provided in a probiotic composition
or may be added to the feed or water provided to the subject.

[0021] Suitably, the substrates of nitric oxide synthase are nitrates or
nitrites. The nitrates and nitrites may be provided in the form of a salt
such as sodium nitrate used in the Examples. Other suitable salts include
calcium nitrate, potassium nitrate, sodium nitrite or other salts of
nitrate or nitrite. The nitrate or nitrite can be provided with the
bacteria in a probiotic composition or alternatively may be provided
separately in the feed or water. The nitrates and nitrites may be
provided as a continuous supplement to the feed or water provided to the
subject. In the Examples the nitrate was provided continuously in the
feed at levels between 1 ppm and 1000 ppm. The nitrates or nitrites may
be provided in feed or water at a concentration between 10 ppm and 10,000
ppm, suitably between 50 ppm and 1,000 ppm, suitably between 75 ppm and
500 ppm, suitably between 90 ppm and 200 ppm.

[0022] Probiotic compositions comprising a bacterium capable of producing
nitric oxide and a substrate for nitric oxide synthase, such as nitrate
or nitrite or a salt thereof are also provided.

[0023] The probiotic compositions may further comprise bacteria not
capable of producing nitric oxide. The probiotic compositions may also
include a pharmaceutically acceptable carrier. A pharmaceutically
acceptable carrier is any carrier suitable for in vivo administration.
Examples of pharmaceutically acceptable carriers suitable for use in the
composition include, but are. not limited to, water, buffered solutions,
glucose solutions, oil-based or bacterial culture fluids.

[0024] Additional components of the compositions may suitably include, for
example, excipients such as stabilizers, preservatives, diluents,
emulsifiers and lubricants. Examples of pharmaceutically acceptable
carriers or diluents include stabilizers such as carbohydrates (e.g.,
sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as
albumin or casein, protein-containing agents such as bovine serum or
.skimmed milk and buffers (e.g.,, phosphate buffer). Especially when such
stabilizers are added to the compositions, the composition is suitable
for freeze-drying or spray-drying. The composition may also be
emulsified. Suitably the composition is formulated for inclusion in feed
or water.

[0025] The bacteria capable of producing nitric oxide and the substrate of
nitric oxide synthase may be administered in any order, at the same time
or as part of a unitary composition. The two components may be
administered such that one is administered before the other with a
difference in administration time of a few minutes, 15 minutes, 30
minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours,
1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks or more. The bacteria may
be provided in dosage forms at regularly scheduled intervals or mixed in
feed or water continuously. The substrate of nitric oxide synthase may be
administered at regularly scheduled intervals or in feed and water
continuously as well. In the Examples, the probiotic bacteria were
administered in a dosage form by oral gavage and the substrate of nitric
oxide synthase was administered continuously in the feed.

[0026] Administration of bacteria capable of producing nitric oxide with a
substrate of nitric oxide synthase to subjects is capable of improving
the health of the subjects after administration. In particular, the
methods provided herein are capable of improving the gastrointestinal
health of the subjects. This may include reducing the incidence or
severity of necrotic enteritis (by at least 10%, 15% or even 20% as
compared to controls), or reducing the bacterial load in the intestines
of the animal, specifically with regards to levels of at least one
pathogenic bacteria. As used herein, pathogenic bacteria include bacteria
capable of causing disease in the subjects or in a human. Disease
includes mortality, morbidity, or reduced productivity of agricultural
animals, e.g., reduced weight gain, reduced offspring, egg or milk
production, or reduced feed conversion ratio. For example the levels of
Salmonella, Campylobacter, E. coli or Clostridium perfringens in the
gastrointestinal tract of animals may be reduced (at least 50% decrease
in recovery, suitably at least 60%, 70%, 80% or even 90% decrease in
recovery as compared to controls). Improving the gastrointestinal health
may also be quantified by an increase in the daily average weight gain of
an animal (at least 3% increase, suitably at least a 5%, 7%, 10%, 20%,
30%, 40% or even 50% increase in weight gain as compared to controls over
a set period of time such as a week or month). A suitable control is a
similar subject not administered bacteria capable of producing nitric
oxide with a substrate of nitric oxide synthase or the subject prior to
administration of the bacteria and the substrate of nitric oxide
synthase.

[0027] The methods may also reduce the level or number of potential
bacterial food-borne pathogens of humans in the gastrointestinal tract of
commercial agricultural animals as compared to controls (at least 50%
decrease in recovery, suitably at least 60%, 70%, 80% or even 90%
decrease in recovery as compared to controls). In particular, the level
of Salmonella and Campylobacter spp. in the gastrointestinal tract of
animals may be reduced in animals administered bacteria capable of
producing nitric oxide and a substrate of nitric oxide synthase.

[0028] Such a reduction in potential human pathogen load in the
gastrointestinal tract of animals will limit the opportunity of
contaminating the human food chain either during preparation of meat for
human consumption or via contamination of animal products such as poultry
eggs. In addition, reduction of the pathogenic bacterial load in animals
treated with, the methods described herein may also reduce horizontal
transmission of pathogenic bacteria within a group of animals (suitably
horizontal transmission is reduced by at least 10%, 20%, 30%, 40% or as
much as 50%). As used herein, pathogenic bacteria include any bacteria
capable of causing morbidity or mortality in the animal being treated
using the methods described herein or in immunocompetent humans.

[0029] Suitably the subjects used in the methods are humans, mammals or
poultry, suitably the animals are domesticated agricultural animals such
as cows, pigs, sheep, or poultry, suitably a chicken or turkey. If
supplied in an animal feed, the feed may comprise between 105 and
108 cfu total bacteria/gm of finished feed. Suitably the feed
comprises between 106 and 107 cfu bacteria/gm feed. The
probiotic formulation or the bacteria capable of producing nitric oxide
and the substrate of nitric oxide synthase may be added to feed during
production, after production by the supplier or by the person feeding the
animals, just prior to providing the feed to the animals. The bacteria
capable of producing nitric oxide and the substrate for nitric oxide
synthase may be provided as a single dosage form, administered
simultaneously, or administered sequentially or completely separately.

[0030] Methods of reducing the inflammatory immune response after
treatment with a probiotic composition are also provided. The probiotic
composition may include bacteria capable of producing nitric oxide or
bacteria capable of producing nitric oxide may be administered in
conjunction with the probiotic composition. Although probiotic
compositions are generally used to increase the health of the animals
being treated as well as reduce the number of pathogenic bacteria in the
gastrointestinal tract of the animals, some probiotic treatments may
induce an inflammatory immune response to the administered probiotic
bacteria which may limit the benefit of the probiotic in terms of body
weight gain. Reduced body weight gain may be associated with inflammatory
immune responses in animals and may reduce the agricultural benefits of
probiotic treatments. Addition of bacteria capable of producing nitric
oxide and a substrate of nitric oxide synthase may reduce inflammation in
the subject to which they are administered and may result in increased
weight gain.

[0031] In the Examples, addition of nitrate to chicken feed at 100 ppm was
shown to decrease the production of inflammatory mediators, specifically
IL-2, IL-4 and IFN-γ. Thus, oral administration of a substrate of
nitric oxide synthase to a subject, in combination with a
NO-production-capable probiotic, may decrease the inflammatory immune
response and increase the overall health of the subject in comparison to
control subjects. Control subjects include subjects treated with a
probiotic alone or untreated subjects. Immune effectors include but are
not limited to cytokines or growth factors such as IL-1, IL-2, IL-4,
IL-6, IL-10, TNF-α, IFN-γ, IFNα/β, TGF-β.

[0034] The feed also contained 0.1% lactose as a prebiotic. The feed was
suspended in 4.5 mL sterile saline and inoculated with 0.5 mL of
Salmonella enteriditis (SE) culture containing approximately 104
cfu/ml. The tubes were treated with either 0.6 ml of 106 cfu/ml of
FloraMax-B11® probiotic or saline as a negative control. After
administering the treatment, the tubes were agitated and incubated at
42° C. for 24 hours. The tubes then were agitated and the content
was serially diluted and plated on Brilliant Green Agar (BGA) containing
novobiocin (250 ml) and nalidixic acid (20 μl/ml) to select for SE.
Typical SE colonies were counted after 24 hours of incubation. The
results are shown in FIG. 1. Addition of increasing amounts of sodium
nitrate resulted in decreased recovery of SE as compared to the control.
In particular, addition of 100 ppm or 1000 ppm of nitrate with the
FloraMax-B11® probiotic reduced the levels of Salmonella recovery
equal to or beyond the reduction with the probiotic alone in the in vitro
assay.

In vivo Testing

Experiment 1

[0035] One-hundred and eighty day-of-hatch chicks were gavaged with
1.75×104 cfu/chick of SE and randomly assigned to a group
(n=30). One hour later chicks were gavaged with either
2.17×107 cfu/chick of FloraMax-B11° probiotic or skim
milk. Nitrate was included in the feed at either 1000 ppm, 100 ppm, 10
ppm, 1 ppm or 0 ppm according to treatment group throughout the testing
period. Chicks were harvested at 24 and 72 hr after administration of the
indicated treatment. Crop and cecal tonsils from 15 chicks/group were
removed and enriched overnight in tetrathionate broth. Then samples were
plated. on BGA containing novobiocin and naladixic acid as described
above.

[0036]FIG. 2 and FIG. 3 show the resulting recovery of SE at 24 and 72
hours after administration. In the graphs different letters within sample
types (i.e., crop or cecal tonsils) indicates significantly different
values (P<0.05). As can be seen. in FIG. 2, administration of the
probiotic alone or probiotic in combination with nitrate had no effect on
the levels of SE recovered from the crop, but did significantly affect
the levels of SE recovered from the cecal tonsils at 24 hours after
administration. Notably the addition of nitrate had no effect at this
time point beyond the probiotic alone. FIG. 3 shows that at 72 hours
after administration, there was again no significant effect on SE
recovery from the crop, but the trend in this preliminary experiment
suggested that the crop levels of SE were slightly reduced after
administration of the probiotic in combination with 100 ppm nitrate.
Conversely, significant effects were observed in the levels of SE
recovery in the cecal tonsils. Administration of the probiotic alone or
in combination with 100 ppm nitrate reduced recovery of SE. While not
statistically significant, the addition of notrate further reduced
recovery of SE as compared to administration of the probiotic alone.
These results suggest that administration of the probiotic reduces
invasion of SE to deeper tissues from the gastrointestinal tract.

Experiment 2

[0037] One-hundred and twenty day-of-hatch chicks were gavaged with
3.0×104 cfu/ chick SE and randomly assigned to a group (n=40).
One hour later chicks were gavaged with either 1.0×107
cfu/chick of FloraMax-B11® probiotic or skim milk. Nitrate was
included in the feed at either 100 ppm or 0 ppm according to treatment
group. Chicks were harvested at 24 and 72 hr. Crop and cecal tonsils from
20 chicks/group were removed and enriched overnight in tetrathionte
broth. Then samples were plated on BGA containing novobiocin and
naladixic acid. Ceca from 10 chicks/group were collected for later mRNA
isolation and analysis of IL-2, IL-4 and IFN-γ levels.

[0038]FIG. 4 and FIG. 5 show the resulting recovery of SE at 24 and 72
hours after administration. In the graphs different letters within sample
types (i.e., crop or cecal tonsils) indicates significantly different
values (P<0.05). As can be seen in FIG. 4, administration of the
probiotic alone or probiotic in combination with nitrate had a
statistically significant effect on the levels of SE recovered from the
crop and the cecal tonsils at 24 hours after administration. No further
improvement in reducing SE recovery was observed when the probiotic was
administered with nitrate. In FIG. 5, administration of the probiotic
alone or in combination with nitrate resulted in reduced SE recovery from
both the crop and the cecal tonsils at 72 hours after administration.
Notably, the addition of nitrate reduced SE recovery from both the crop
and cecal tonsils at 72 hours post-administration. Thus, administration
of the probiotic in combination with nitrate may result in a further
increase in reduction of SE recovery particularly at later times after
administration.

[0039] The results of the mRNA analysis of inflammatory mediators IL-4,
IFN-γ and IL-2 are shown in FIGS. 6-8, respectively. FIGS. 6-8
shows that the levels of IL-4, IFN-γ and IL-2 are increased after
administration of the probiotic as compared, to the control and that
IL-4, IFN-γ and IL-2 levels are decreased after administration of
the probiotic in combination with nitrate. These results suggest that the
co-administration of the probiotic with nitrate results in a decreased
inflammatory immune response to the bacteria in the probiotic.
Inflammation is associated with decreased weight gain and reduced feed
conversion ratios in agricultural animals.

Patent applications by Amanda Wolfenden, Pea Ridge, AR US

Patent applications by Billy Hargis, Fayetteville, AR US

Patent applications by The Board of Trustees of the University of Arkansas

Patent applications in class Intentional mixture of two or more micro-organisms, cells, or viruses of different genera

Patent applications in all subclasses Intentional mixture of two or more micro-organisms, cells, or viruses of different genera